The elementary unit of Ca2+ release in heart muscle is the calcium spark, discovered by the PI in 1993. The molecular mechanisms that underlie activation, modulation and termination of Ca2+ sparks remain elusive or controversial. The proposed work will use new methods developed by the PI to investigate the biophysical properties of Ca2+ sparks that should enable us to broaden our understanding of cardiac Ca2+ signaling and resolve the conflicts. The proposed patch clamp experiments will use cardiac myocytes from rats and rabbits to examine Ca2+ sparks. Simultaneous confocal calcium imaging and flash photolysis of caged Ca2+ will permit a quantitatively investigation of Ca2+ spark behavior. Sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors or RyR2s) will be studied in parallel experiments using a planar lipid bilayer technique with flash photolysis and rapid solution switching methods. Preliminary results suggest that all proposed experiments can be done and should provide important new information. There are four questions that the proposed work seeks to address. 1 How does the triggering of Ca2+ sparks depend on [Ca2+]i and important cellular regulators of EC coupling? 2. How does RyR2 behavior influence the triggering of Ca2+ sparks? 3. How does membrane potential influence Ca2+ spark triggering? 4. What mechanisms underlie Ca2+ spark restitution? The planned work will provide fundamental new information on how Ca2+ sparks work in heart. It will examine Ca2+-induced Ca2+-release (CICR), the triggering by [Ca2+]i of Ca2+ sparks, the regulation of CICR and Ca2+ spark behavior and how restitution of the Ca2+ spark occurs. It will also link Ca2+ spark properties to the behavior of RyR2s. The proposed work should broaden our understanding of cardiac Ca2+ signaling, excitation-contraction (EC) coupling and spontaneous SR Ca2+ release. This investigation is part of the Pl's long-term goal to understand how normal physiological signaling regulates heart muscle and how molecular and cellular alterations of such signaling systems lead to specific pathologies and novel treatments.